Gene therapy for ocular disorders is reaching the clinic, with long-term trials of gene therapies for Leber's congenital amaurosis reporting improvements in patients (Bainbridge, Mehat et al. 2015; Jacobson, Cideciyan et al. 2015). Promoters such as the chicken beta actin and cytomegalovirus are common in current gene therapies, particularly because they are strong, small, and well characterized. However, in applications where restricted expression is desired, such as targeting transcription to a specific tissue or limiting it to particular cells, a toolbox of specific promoters would be advantageous. Previously, we have developed mini-promoters (approximately four kb human regulatory sequences for tissue and cell-specific expression), using bioinformatics, and single copy knock-into the mouse genome (Portales-Casamar, Swanson et al. 2010; de Leeuw, Dyka et al. 2014). Building on, and further refining these techniques, we are expanding the current toolbox by introducing new mini-promoters from PAX6 (paired box 6 (OMIM: 607108)).
Although PAX6 is expressed in a variety of tissues including the CNS, pancreas, and small intestine, it is best known as the essential transcription factor for panocular development in species as diverse as flies (Drosophila melanogaster), mice (Mus musculus), and humans, see Cvekl et al. 2016 for a review (Cvekl and Callaerts 2016). In humans, loss-of-function mutations produce the ocular disorder aniridia (OMIM: 106210). Current interventions can delay blindness due to aniridia, but new therapies are needed to safe guard or even restore patient vision, and gene therapy is one promising therapeutic approach.
One challenge for PAX6 gene therapy is that transcription of the endogenous gene is complex, and inappropriate PAX6 could be detrimental. Ectopic expression of PAX6 orthologues in D. melanogaster and Xenopus laevis resulted in the formation of ectopic eyes (Halder, Callaerts et al. 1995; Chow, Altmann et al. 1999). Furthermore, overexpression of PAX6 in mice interfered with ocular development resulting in microphthalmia (Schedl, Ross et al. 1996; Manuel, Pratt et al. 2008). Finally, expression is temporally regulated with, for example, broad and robust developmental expression being restricted to ganglion, amacrine, horizontal, and Müller glia cells in the adult retina. At least 39 cis-regulatory elements have been verified in vivo, see Cvekl et al. 2016 for a review (Cvekl and Callaerts 2016). Of these elements, those with known adult expression, and those amenable to “cut down” are of greatest interest, as they would be suited for gene therapies administered after development, and enable packing in the size restricted 4.9-kb rAAV (recombinant adeno-associated virus) genome.
There is a need for characterized human PAX6 promoters for gene expression, for instance in human gene therapy applications. It is in particular useful to identify small promoter elements that are sufficient to drive expression in certain cell types, for instance retinal cells. Such small promoter elements, or “mini-promoters” are particularly useful in certain applications, for instance they are more amenable to insertion into viral vectors used in gene therapy applications.